spontaneous conversion g PrP g PrP C g PrP g PrP C non-seeded Supplementary Figure 1. Determining the time-span of qRT-QuIC. RT-QuIC reactions were seeded with PrP or PrP C with the indicated amounts. A non-seeded reaction was performed as the control. After 130 h at 37 o C, both PrP C -seeded and non-seeded reactions showed rising curves, indicating that the spontaneous conversion started at approx. 100 h. Therefore, we chose 90 hours as the maximum time-span of monitoring prion conversion in the qRT-QuIC system.

PrP PrP C RML gRML g RML g RML g RML g RML g RML g RML g RML g RML g RML g RML g Supplementary fig 2 3 h 4 h 5 h 7 h 10 h 12 h 16 h 26 h 46 h 56 h 71 h76 h 0 h30 h60 h90 h RFU 0 h30 h60 h90 h

3 h 5 h 7 h 8 h PrP PrP C ME g ME g 9 h 10 h 15 h 30 h 38 h 50 h 62 h 85 h ME g ME g ME g ME g ME gME g ME g ME g ME gME g RFU 0 h30 h60 h 90 h0 h30 h60 h 90 h Supplementary Figure 2 and 3. Detecting seeded PrP with RT-QuIC. Purified RML and ME7 PrP and control PrP C with the quantities from to g were seeded into reactions independently to perform 90 h of RT-QuIC at 37 o C.

RT-QuIC process in hours number of positive RT-QuIC reaction RT-QuIC seeded with RML PrP (10 y g) number of positive RT-QuIC reaction RT-QuIC process in hours RT-QuIC seeded with ME7 PrP (10 y g) Supplementary Figure 4. The positive RT-QuIC reactions seeded with PrP ( to g for RML and to g for ME7) within 90 h are shown. Each scale on the Y-axis represents one effective reaction, the X-axis indicates the required hours corresponding to the reaction. The reactions seeded with g of RML PrP were negative up to 90 h and thus are not shown in the figure.

PrP concentration (×10 -5 g/g brain) with quantitative RT-QuIC (qRT-QuIC) with quantitative immunoblot (qIB) RML 90 dpi1.16± ± dpi2.19± ± dpi5.51± ± dpi6.83± ± dpi ±0.75 ME7 90 dpi0.75± ± dpi1.44± ± dpi3.20± ± dpi3.77± ±0.19 Supplementary Table 1. Comparing PrP concentrations measured by quantitative RT-QuIC and quantitative immunoblot

PrP concentration in tissues (g/g brain) brainheartliverspleenlungkidneymuscle 30 dpi (9.05±1.48) × Neg. (1.42±0.25) × (1.98±0.52) × Neg. (4.50±0.92) × Neg. 60 dpi (5.21±1.61) ×10 -9 Neg. * (6.07±0.45) × (1.05±0.41) × Neg. (1.89±0.27) × Neg. 90 dpi (1.16±0.44) ×10 -5 Neg. (6.10±0.73) × Neg. 120 dpi (2.19±0.38) ×10 -5 Neg. (4.78±0.82) × Neg. (1.62±0.17) × dpi (5.52±1.44) ×10 -5 * (5.86±0.27) × Neg. (4.60±1.24) ×10 -9 Neg. (1.73±0.35) × dpi (6.83±1.01) ×10 -5 (1.64±0.09) × * (1.13±0.18) × (1.03±0.15) ×10 -8 Neg. * (5.38±0.39) × (1.31±0.64) × dpi7.33×10 -5 (1.07±0.22) × (2.95±0.57) × (6.42±1.77) ×10 -9 Neg. (1.42±0.16) × (1.63±0.33) ×10 -9 Supplementary Table 2. The concentration of PrP in 7 tissues from RML scrapie-infected mice of 7 time-points Neg indicates the RT-QuIC reaction is negative up to 90 hours. The mean and s.e.m are shown. * The concentration of PrP is lower than one LD 50 unit per gram of brain (1.16 × g/g brain).

PrP concentration in tissues (g/g brain) brainheartliverspleenlungkidneymuscle 30 dpi (3.64±1.97) ×10 -8 Neg. (2.20±1.06) ×10 -8 Neg. 60 dpi (9.01±2.31) ×10 -8 Neg. (1.34±1.14) ×10 -8 Neg. 90 dpi (7.96±0.78) ×10 -6 Neg. (8.20±5.79) ×10 -8 Neg. (1.77±1.16) × dpi (1.51±0.80) ×10 -5 (4.90±4.62) × Neg. (6.56±3.48) ×10 -8 Neg. (1.18±1.00) × dpi (3.56±1.88) ×10 -5 (1.75±1.44) × (2.67±1.45) × (9.57±1.70) ×10 -8 Neg. * (6.45±2.92) × (2.09±0.79) × dpi (3.77±1.55) ×10 -5 (2.23±1.14) × (3.20±1.85) × (7.48±3.76) ×10 -8 Neg. * (9.43±3.00) × (2.34±0.98) ×10 -8 Neg indicates the RT-QuIC reaction is negative up to 90 hours. The mean and s.d. are shown. * The concentration of ME7 PrP is lower than its one LD 50 unit per gram of brain (1.89× g/g brain). Supplementary Table 3. The concentration of PrP in 7 tissues from ME7 scrapie-infected mice of 6 time-points